PROJECT SUMMARY/ABSTRACT Events in early implantation are key to fertility and pregnancy success, and the uterine lining (endometrium) plays a critical role in assuring acceptance and sustenance of the conceptus at the beginning and throughout gestation. Essential for pregnancy establishment and maintenance is the progesterone (P4)-induced differentiation (decidualization) of the endometrial stromal fibroblast (eSF) to the ?decidualized? phenotype, characterized by major genomic, transcriptomic, morphologic and functional transformations. We found that human eSF derive from the endometrial mesenchymal stem cell (eMSC), and in women with endometriosis, an inflammatory disorder associated with infertility, miscarriage and other poor pregnancy outcomes, eSF have an abnormal response to P4 that is inherited from eMSC, compared to controls. We also found that steroid hormones regulate the eSF DNA methylome and women with endometriosis have pre-existing eSF genome- wide aberrant epigenetic marks associated with gene expression changes and that their eSF have a pro- inflammatory phenotype likely acquired in the endometrial niche. Our overall hypothesis is that epigenetic mechanisms regulate eMSC differentiation and the decidualization of their eSF progeny and that inflammation in endometriosis alters these processes and compromises eSF function and the steroid hormone response essential for successful implantation. We propose 3 specific aims: Aim 1: Test the hypothesis that genome- wide epigenetic programming of eSF is inherited from its progenitor and changes in response to steroid hormones and affects eSF functionality. Aim 2. Determine endometrial immune features of the inflammatory milieu in endometriosis and their effects on eMSC lineage differentiation and eSF decidualization. Aim 3. Test the hypothesis that endometriosis results in an aberrant eSF response to soluble signals from placental cytotrophoblasts (CTB) important in implantation. We propose advanced molecular and epigenomic approaches, functional studies to determine roles of candidate genes in decidualization, innovative CyTOF technology to deeply phenotype specific immune populations in the endometrium, advanced computational methods for data integration, and deep learning tools combined with high-throughput imaging to map changes in cell morphology space onto transcriptome changes in epigenetically regulated cells. Understanding mechanisms underlying eSF lineage specification from eMSC, eSF response to steroid hormones, communication with the invasive CTB, and effects of inflammation on these processes is highly significant to reproductive success and the health and well-being of those who conceive and their offspring. The expected results are to derive an in-depth understanding of the role of P4-resistance and inflammation in endometriosis that can also inform infertility and poor pregnancy outcomes in women with other inflammatory disorders. Also, reprogramming eSF and eMSC could lead to novel therapies to maximize functionality of these cells in normal implantation and endometrial homeostasis more broadly.